Cell culture incubator

ABSTRACT

A cell culture incubator has a chamber divided into an incubation portion and a control portion. The incubation portion is adapted to contain a cell culture receptacle. A cell culture receptacle agitator is located in the control portion of the incubator and linkage between the cell culture receptacle agitator and the incubation portion of the chamber causes agitation of the cell culture receptacle. A heater in the control portion of the chamber heats air from the external environment and transports the heated air to the incubation portion of the chamber. A wall divides the chamber into the incubation portion and the control portion. The heater may be a forced air heater that provides a positive pressure to the incubation portion of the chamber to reduce contamination in the incubation portion. A nutrient and gas circulation system communicates with the cell culture receptacle in the incubation portion of the chamber. The nutrient and gas circulation system includes a gas source external to the incubation portion of the chamber and tubing provides communication between the gas source, the cell culture receptacle in the incubation portion of the chamber, and nutrient media. A filter in an opening of the incubation portion of the chamber communicates with the tubing to remove contaminants from gas emanating from the cell culture receptacle prior to venting to the external environment.

STATEMENT OF RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 08/740,729 filed Nov.1, 1996, which is in turn a continuation-in-part of U.S. Ser. No.08/512,546 filed Aug. 8, 1995 now U.S. Pat. No. 5,622,857, which is inturn a continuation-in-part of PCT/US94/02140 filed Feb. 9, 1994.

FIELD OF THE INVENTION

This invention relates to an incubator instrument for the operation ofperfusion cell culture processes on the laboratory bench without anyspecial sterility or biohazard features in the working environment. Inaddition, this invention provides the means for mixing both perfusionand non-perfusion based cell culture processes. Furthermore, the meansfor achieving a controlled CO₂ atmosphere and the required temperature(e.g., at 37° C.) is provided for perfusion, mixed and static cultures.Additionally, the invention relates to the use of poly anionic mediaadditives which enhance attachment of adherent cells to microcarrierswhile mixing at relatively high speeds, and provide other benefits byfacilitating in vivo-like conditions in perfusion culture systems.

BACKGROUND OF THE INVENTION

The standard practice in cell culture is to employ a CO₂ incubator toprovide the gaseous atmosphere and temperature control required instatic culture processes. These processes include the use of multiwellplates, culture dishes, flasks and the like. The relatively large sizeof standard CO₂ incubators (with an inside working volume of about 19in. wide×26 in. high×19 in. deep), has been used to accommodate smallscale processes employing perfusion and mixing equipment, such as pumpsand roller mills. Besides being highly inconvenient, such use of CO₂incubators require substantial capital expenditures and spaceutilization.

From a process efficiency standpoint, perfusion cell culture processesin which O₂ /CO₂ containing gases are provided to the cultured cells bya direct means are the methods of choice. Various costly, cumbersome,and complex instrument systems have been developed to operate perfusionand mix/stirred culture systems. While performance improvements can beachieved with such systems, they are invariably dedicated and ratherinflexible instruments. Also, the failure rate of these stand aloneinstruments tend to be relatively high. The primary cause of failure isa malfunction of the hot plate which is typically employed for heatingthe media reservoir. Reports from users of the heater over shooting theset point and an inability to accurately control the temperature inlong-term experiments are not uncommon. Most important of all, theinstruments are extremely user unfriendly; often requiring years ofexperience in order to competently conduct a series of completeprocedures without the intervention of a technical expert.

The creation of in vivo-like conditions in cell culture systems is along-standing challenge. Perfusion and mixed/stirred systems areparticularly problematic because cells are exposed to shared forces andother effects related to motion which are not normally experienced invivo. While attachment factors and extracellular matrix components havebecome increasing employed in the last 20 years, they remain a researchcuriosity primarily due to economic reasons. An inexpensive cell culturemedia additive is required which has general applicability. Such amolecule should fulfill the following three requirements:

(i) enhance cell adhesion in a non-selective way, such thatmixing/stirring can proceed efficiently in large scale culture systems;

(ii) manipulations of cells in culture (e.g., genetic transformations)must proceed unimpeded; and,

(iii) culture conditions approaching the cellular environment found intissues must be advanced.

Definitions

CO₂ --carbon dioxide gas for maintaining pH, in combination withbicarbonate buffer in the cell culture nutrient media.

Incubator--an instrument designed to provide the culture environment forcells.

O₂ --oxygen gas, necessary for biological cells to respire.

HPBr--high performance (hollow fiber) bioreactor; a perfusion cellculture device having, for example, a central bi-directional hollowfiber bundle that supplies media, and an outer fiber bundle area thatsupplies oxygen needed for cell culture.

cpm--cycles per minute, which is the number of 120 degree (for example)complete bi-directional partial rotations of the device in the period ofa minute.

Lipofection--introduction of foreign DNA into a host cell which ismediated by cationic lipids that form positively charged liposomes.

Transfection--a process whereby foreign DNA, which is not capable ofintegrating into the host cell's genome, is introduced into the hostcell.

SUMMARY OF THE INVENTION

A cell culture incubator has a chamber divided into an incubationportion and a control portion. The incubation portion is adapted tocontain a cell culture receptacle. A cell culture receptacle agitator islocated in the control portion of the incubator and linkage between thecell culture receptacle agitator and the incubation portion of thechamber causes agitation of the cell culture receptacle. A heater in thecontrol portion of the chamber heats air from the external environmentand transports the heated air to the incubation portion of the chamber.A wall divides the chamber into the incubation portion and the controlportion. The heater may be a forced air heater that provides a positivepressure to the incubation portion of the chamber to reducecontamination in the incubation portion. A nutrient and gas circulationsystem communicates with the cell culture receptacle in the incubationportion of the chamber. The nutrient and gas circulation system includesa gas source external to the incubation portion of the chamber andtubing provides communication between the gas source, the cell culturereceptacle in the incubation portion of the chamber, and nutrient media.A filter in an opening of the incubation portion of the chambercommunicates with the tubing to remove contaminants from gas emanatingfrom the cell culture receptacle prior to venting to the externalenvironment.

The invention provides a small footprint benchtop incubator instrumentdesigned to accommodate perfusion mixed and static cell cultureprocesses. To accommodate perfusion processes, the invention is equippedwith a peristaltic pump for use with sterile disposable tubing sets ofvarying configurations. The tubing sets provide the means for circulatenutrient media from a reservoir. O₂ /CO₂ containing gases are suppliedfrom a premixed gas cylinder, and sterility/biohazard concerns areaddressed by a series of strategically placed 0.22 μm hydrophobic discfilters. The tubing set design enables a closed loop recirculation ofgases and, optionally, an auxiliary supply of the defined gas mixturecan be delivered to a disposable flow through chamber which can containculture plates, flasks and the like. Alternatively, the gas flow tubingset can be modified to supply sterial defined O₂ /CO₂ gas mixtures toroller bottle cultures and the like. Temperature is maintained in theworking volume of the incubator by thermostatically controlledrecirculating air, which is maintained at positive pressure relative toambient conditions. The invention further provides a means for mixingeither a perfusion or other batch culture vessels, such as a cultureflask or bottle. Sufficient space is provided in the working volume ofthe incubator such that a reasonable number of culture dishes or flaskscan be cultured under identical conditions, which may be critical inprocess development and scale-up activities. Thus, controls or eveninoculum expansion can be cultured in parallel.

In addition, the invention provides for the use of chondroitin sulfate(type C) as a cell culture media additive to enhance adhesion ofanchorage dependent cells to microcarriers while mixing and stirring.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same becomesbetter understood by reference to the following detailed description,when taken in conjunction with the accompanying drawings, wherein:

FIG. 1 is a perspective view, partially exposed, showing the cellculture incubator of the present invention;

FIG. 2 is a perspective view of a first modular component of the chamberof the cell culture incubator of the present invention;

FIG. 3 is a perspective view of a second modular component of thechamber of the cell culture incubator of the present invention;

FIG. 4 is a perspective view of a third modular component of the chamberof the cell culture incubator of the present invention;

FIG. 5 is a perspective view of a fourth modular component of thechamber of the cell culture incubator of the present invention;

FIG. 6 is an exploded perspective view of the interrelationship betweenthe four components of FIGS. 2 through 5 that form the chamber of thecell culture incubator of the present invention;

FIG. 7 is an exposed front view of the cell culture incubator of thepresent invention;

FIG. 8 is an exposed end view of the cell culture incubator of thepresent invention;

FIG. 9 is an exposed view of the cell culture incubator of the presentinvention;

FIG. 10 is a detailed side view of the agitator assembly of the cellculture incubator of the present invention;

FIG. 10A is a detailed view of the crank, cam, and the rod of theagitator assembly of the cell culture incubator of the presentinvention;

FIG. 11 is a schematic view of the nutrient and gas circulation system,continuous-batch process, for the cell culture incubator of the presentinvention;

FIG. 12 is a schematic view of the nutrient and gas circulation system,continuous process, for the cell culture incubator of the presentinvention;

FIG. 13 is a schematic view of the nutrient and gas circulation system,auxiliary gas supply, for the cell culture incubator of the presentinvention;

FIG. 14 is a graph of the effect of cell agitation in the cell cultureincubator of the present invention on cumulative product of IgG₁monoclonal antibody by 3C11 hybridoma cells; and

FIG. 15 is a bar graph of the effect of cell agitation in the cellculture incubator of the present invention on 3C11 hybridoma cellviability and fold increase.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As shown in FIG. 1, the cell culture incubator of the present inventionis comprised of a chamber 2 which is divided into an incubation portion4 and a control portion 6 by wall 8. As best shown in FIGS. 2 through 6,chamber 2 is comprised of four modular components 10, 12, 14, and 16.Modular components 10, 12, 14, and 16 are preferably comprised of asynthetic polymer such as acrylic or the like and are preferably joinedtogether by mating gaskets such that the bearing weight of modularcomponents 10, 12, 14, and 16 joins them. As shown in FIG. 6, thejoining of modular components 10, 12, 14, and 16 form chamber 2 of thecell culture incubator of the present invention in an economical andconvenient manner.

Referring to FIGS. 1, 7, 8, and 9, chamber 2 of the cell cultureincubator of the present invention includes four basic systems whichoptimally facilitate incubation of cell cultures: Temperature RegulationSystem, Media Circulation System, Gas Flow System, and Culture AgitationSystem.

Temperature Regulation System

The Temperature Regulation System includes air inlet 18 which allows airfrom the external environment to enter control portion 6 of chamber 2.Optimally, if improved sterility is desired, a filter 19 of, forexample, 0.22 μm porosity can be placed adjacent air inlet 18 to ensurethat ambient air entering control portion 6 of chamber 2 does notcontain contaminants. Once air enters control portion 6 of chamber 2through air inlet 18, it passes into hot box 20 through hot box inlets22. Located within hot box 20 is a forced air heater 21 that increasesthe thermodynamic energy of the air in hot box 20. Heater 21, preferablymounted in wall 8, forces the heated air through wall 8, and then intoincubation portion 4 where cell culture receptacle 28 (such as a HPBrhollow fiber device) is located. In order to maintain equilibrium, aportion of the now cooled air that was first heated and forced intoincubation portion 4 from control portion 6 is recycled to hot box 20 incontrol portion 6 through conduits 23 that connect return openings 24 inhot box 20 to incubator portion air outlets 26 in wall 8. Heated air inincubation portion 4 that does not return to control portion 6 throughincubation portion air outlet 26, is expelled into to the externalenvironment through interstices between modular components 10, 12, 14,and 16. Thus, a substantially open, laminar flow system with minimalpressure build-up is provided. In this manner, rapid temperatureresponses combined with accurate and stable temperature control,relative to the set point, are achieved. Preferably, the slightlypositive pressure in the incubation portion 4 of chamber 2 relative toambient pressure reduces the possibility of contaminants from ambientair entering incubation portion 4 of chamber 2. In order to ensure theaforesaid accurate and stable temperature control, a temperaturecontroller 29 such as Model No. CN76120 manufactured by OmegaEngineering Incorporated of Stamford, Conn., which is a one-pulse outputmicroprocessor with an alarm, may be employed. Additionally, locatedwithin incubation portion 4 of chamber 2 there is preferably located athermocouple 30, for example, Model No. 5TC-TT-T-24-36 manufactured byOmega Engineering Incorporated of Stamford, Conn., which providesfeedback to the above temperature controller 29 and is preferablyinsulated with, for example, "TEFLON" or the like.

In summary, the above temperature regulation system allows the desiredthermodynamic energy to be provided for incubation portion 6 whilemaintaining a cooler environment in control portion 4 such that theelectrical and mechanical components therein are not subject tothermodynamic-based fatigue and damage.

Culture Agitation System

The Culture Agitation System of the present invention, as best shown inFIGS. 1, 7, 9, 10, and 10A includes motor 34 located on motor mount 36and having drive rod 38 passing through motor mount 36. Drive rod 38 isfixedly connected to crank 40, and crank 40 is connected to cam 42 bytie rod 44, as shown in FIG. 10A. It is to be noted that tie rod 44 ispivotally connected to both crank 40 and cam 42 at pivot points 41 and43 that are offset from the axes of rotation of crank 40 and cam 42,respectively, such that 360 degree rotation of drive rod 38 and crank 40causes a bi-directional partial rotation of about, for example, 120degree of cam 42. Cam 42 is fixedly attached to axle 46, which issupported by axle mounts 48. It is important to note that all of theabove elements are entirely located within the relatively cooler controlportion 6 of chamber 2, with the exception of axle 46 which spans bothcontrol portion 6 of chamber 2 and incubation portion 4 of chamber 2,and some of axle mounts 48 which are located within incubation portion 4of chamber 2. Also located within incubation portion 4 of chamber 2 arecradle supports 50 which connect cradle 52 to axle 46. In cradle 52' islocated cell culture receptacle 28. In operation, the 360 degreerotation of drive rod 38 and crank 40, which is translated toapproximately 120 degree bi-directional partial rotation of cam 42 andaxle 46 by the interconnection of cam 42 and crank 40 by tie rod 44,causes approximately 120 degree bi-directional partial rotation ofcradle 52 and cell culture receptacle 28 to effectuate the appropriatecell culture agitation within the cell culture receptacle 28. Motor 34preferably has four settings; static, low (12±3 cpm), medium (30±3 cpm),and high (60±3 cpm).

While the above embodiment employs translation of 360 degree rotation to120 degree bi-directional partial rotation, partial rotations of otherdegree components could be employed, as well as total 360 degreerotation, axial reciprocation, vertical reciprocation, or a combinationof the above.

During cell culture agitation, it is often desirable to employ proactivemedia components in the cell culture. Preferably, chondroitin sulfate(Type C) is employed as a media component when anchorage dependent cellsare cultured in order to enhance attachment to the carrier surface(e.g., microcarriers) during agitation. The preferred concentrationrange of chondroitin sulfate (Type C) is molecular weight dependent.However, at a mean molecular weight of about 4,000 daltons, thepreferred concentration is in the range of about 0.005 mM to 0.5 mM.

Media Circulation System and Gas Flow System

The Media Circulation System and the Gas Flow System are two separateand discrete systems of the present invention, however they will bediscussed in tandem for the sake of clarity with the divisions beingbased upon the type of process being employed or the presence ofauxiliary functions as shown in FIGS. 11, 12, and 13. For example, FIG.11 shows both the Media Circulation System and Gas Flow System used in acontinuous-batch process, FIG. 12 shows the Media Circulation System andGas Flow System in a continuous process and FIG. 13 shows the MediaCirculation System and Gas Flow System employing an auxiliary gassupply.

First, referring to FIG. 11, which shows the Media Circulation Systemand Gas Flow System for the present invention employing acontinuous-batch process, the Gas Flow System includes a gas source 54,for example about 10% CO₂ in air having a pressure valve 56 andsupplying the pressurized gas at about 5 pounds per square inch, forexample. Next, in the Gas Flow System, the pressurized gas passesthrough flow meter 58 which may be, for example, Model No. H-32013-01, a50 milliliter per minute aluminum and stainless steel flow metermanufactured by Cole Parmer Instrument Company, Vernon Hills, Ill. Anoptional check valve and an optional flow restrictor (as shown in FIG.12) set the back pressure of gas in cell culture receptacle 28 inconjunction with flow meter 58. When employed, the check valve isselected to prevent a maximum pressure of, for example, about 0.5 to 1.0psi. from being exceeded. Prior to entering cell culture receptacle 28,the gas passes through pressure gauge 60 to measure the back pressure inthe cell culture receptacle 28, and then flows through hydrophobicfilter 62, which is preferably a filter having a porosity of about 0.22μm. After entering inlet 64 and infusing cell culture receptacle 28, thegas flows out of outlet 66 and through back pressure valve 68 andcondenser 70. Condenser 70 is preferably a coiled section of the plastictubing which preferably forms the lines of the Gas Flow System and theMedia Circulation System. Most preferably, condenser 70 contains glassor plastic beads or particles to maximize the condensation of watervapor entrained by the gas in the cell culture receptacle 28. Next, thegas passes through another hydrophobic filter 72 having a preferredporosity of about 0.22 μm and then enters the head space of mediareservoir 74; in this manner the preferably CO₂ containing gas maintainsthe required pH of the media. The gas is then expelled from theincubation portion 4 of chamber 2 through a gas vent orifice therein andinto the external environment after passing through hydrophobic filter75, preferably having a porosity of about 0.22 μm.

In the Media Circulation System of the continuous-batch process, mediafrom media reservoir 74 passes through pump 76, which is preferably aperistaltic pump Model No. 15PB with a 200 cycle per minute motormanufactured by Barnat Company, Gilmont Instrument, Barrington, Ill.After passing through pump 76, the media enters inlet 78 of cell culturereceptacle 28 where nutrients are provided to the culture. The spentmedia then passes through outlet 80 of cell culture receptacle 28 whereit returns to media reservoir 74. Periodic changes of media reservoir 74in a long-term cell culture procedure defines this process as acontinuous-batch process

Referring to FIG. 12, which shows the Media Circulation System and GasFlow System for the present invention employing a continuous process,the gas flow system includes gas source 82 (for example, about 10% CO₂in air at about 5 pounds per square inch), which communicates with flowmeter 84 (for example, Model No. H-32013-01, a 50 milliliter per minutealuminum and stainless steel flow meter manufactured by Cole ParmerInstrument Company, Vernon Hills, Ill.). Hydrophobic filter 86, whichpreferably has a porosity of about 0.22 μm receives the gas from flowmeter 84. Next, the gas flows through check valve 88 which, inconjunction with flow meter 84 and gas flow restrictor 94 (describedlater) sets the back pressure of gas in cell culture receptacle 28 tothe predetermined level. Check valve 88 is selected to prevent a maximumpressure of, for example, about 0.5 to 1.0 psi from being exceeded.After entering inlet 90 and infusing cell culture receptacle 28, the gasflows out of outlet 92, through flow restrictor 94, and into condenser96. Condenser 96 is preferably a coiled section of the plastic tubing,preferrably made from the same material as the plastic tubing, whichpreferably forms the lines of the Gas Flow System and the MediaCirculation System. Most preferably, condenser 96 contains glass orplastic beads or particles to maximize the condensation of water vaporentrained by the gas in the cell culture receptacle 28. The gas thenflows into the head space of media reservoir 98; in this manner, thepreferably CO₂ -containing gas maintains the required pH of the media.The gas is then expelled from the incubation portion 4 of chamber 2through a gas vent orifice therein and into the external environmentafter passing through hydrophobic filter 100, preferably having aporosity of about 0.22 μm.

In the Media Circulation System of the continuous process, fresh mediais infused into media reservoir 98 from pump 102 while spent media frommedia reservoir 98 is discharged into the external environment throughline 104. The amount of spent media passing through line 104 is directlyproportional to the amount of fresh media being pumped into the closedsystem of media reservoir 98 by pump 102. Pump 102 is preferably aperistaltic pump Model No. 15PB with a 200 cycle per minute motormanufactured by Barnat Company, Gilmont Instrument, Barrington, Ill. Oflike manufacture is pump 106 which takes up media from media reservoir98 such that the media then enters inlet 108 of cell culture receptacle28 where nutrients are provided to the culture. The spent media thenpasses through outlet 110 of cell culture receptacle 28 where it returnsto media reservoir 98 to then pass through line 104 for discharge intothe external environment.

Referring to FIG. 13, which shows the Media Circulation System and GasFlow System for the present invention employing an auxiliary gas supply,like reference numbers used in FIG. 13 that are the same referencenumbers used in FIG. 12 refer to the same elements as shown in FIG. 12.The embodiment of FIG. 13, in which an auxiliary gas supply is employed,differs from the continuous process embodiment in FIG. 12 in that pump102, which supplies fresh media, and line 104, which discharges spentmedia to the external environment, and which are interconnected to mediareservoir 98 in the continuous process embodiment of FIG. 12, are notpresent in the auxiliary gas supply embodiment of FIG. 13. Instead, inthe auxiliary gas supply embodiment of FIG. 13, shunt 112 diverts aportion of the gas flow from condenser 96 that would enter mediareservoir 98 and, instead, directs this portion of gas into flow throughchamber 114, which is designed to contain static culture vessels such asculture plates, for example. After the gas has infused the cultures inflow through chamber 114, it exits chamber 114 through shunt 116 whichjoins the gas flow line from media reservoir 98 that ultimately vents tothe external environment after first passing through hydrophobic filter100.

Culture Examples

A sterile tubing set consisting of the Gas Flow System lines and MediaCirculation System lines is assembled and integrated with the nutrientmedia reservoir 74, 98, employing standard sterile procedures in abiological hood. Alcohol swabs are placed over all fittings which willbe connected to corresponding instrument fittings on the laboratorybench under ambient conditions. The entire assembly is transported tothe cell culture incubator of the present invention. Alcohol swabs areemployed to sterilize the instrument fittings before expeditiouslyremoving the alcohol swabs from the tubing set fittings and completingthe connections in a logical sequence.

Instrument settings (i.e., media circulation rate and gas flow rates)are selected and the tubing set leak tested for a period of time (e.g.,8-24 hours). To minimize cost, phosphate buffered saline (PBS) isrecommended instead of media at this stage. However, this decisiondictates that the instrument must be disassembled and the content of themedia reservoir bottle changed before repeating the above steps andproceeding. Due to the relatively user friendly operational procedure,the cost savings will often more than justify the effort, with nosignificant risk of contamination. The gas source 54, 82 can be changedat any time. The recommended compositional range for most cell cultureprocedures is between 5-10% CO₂ /air. The media circulation should bediscontinued when gas flow is turned off to avoid flooding theoxygenator fibers in the cell culture receptacle 28, if an HPBr device.The present invention will maintain long-term culture with only periodicoperator intervention (e.g., to take samples or to change spent media).

Both cells and microcarriers (for attachment of anchorage dependentcells) are introduced into the cell culture receptacle 28 by ahypodermic needle syringe. Similarly, cells and supernatant (i.e., usedmedia) samples can be removed periodically from the cell culturereceptacle 28 by displacement into an empty syringe with fresh mediafrom a second syringe. While sampling with the door of the incubationportion 4 of chamber 2 open, the temperature controller 29 is switchedoff.

In order to periodically change spent media, the tubing set isdisconnected and alcohol swabs placed over all fittings which have beendisconnected. Under a biological hood, the media is exchanged and thelong-term cell culture process resumed without delay.

EXAMPLE I

The invention employed an HPBr device as cell culture receptacle 28 andwas assembled according to the configuration in FIG. 11. The cellculture receptacle 28 was inoculated with 3.5×10⁸ viable 3C11 hybridomacells and cultured for between 12-15 days in the following cell culturemedia: DMEM containing 4 mM L-glutamine, 1 mM glutamic acid, 2.5 mMbenzoate buffer (comprised of equimolar ratio of benzoic acid and sodiumbenzoate), and 20% fetal bovine serum. Two liters of this media was usedbetween day 1-7, which was replaced with fresh media on day 7. A pH ofbetween about 7.0-7.4 was maintained through out the experiment andmedia was recirculated at 100 mL/min. A 10% CO₂ /air gas mixture wasused at a flow rate of 63 mL(stp)/min and a back pressure of <1.0 psiwas maintained for the duration of the experiment.

Four cell culture receptacles 28 were assemble in this manner, eachusing the following constant rotational speed through out the 12-15 dayperiod: 1) static; 2) 12.sup.± 3 cpm; 3) 30.sup.± 3 cpm; and 4) 60.sup.±3 cpm. After allowing the cells to settle for 1 hour (every week day),10 mL of supernatant was collected from the 17 mL volume within whichthe cells were cultured under perfusion conditions. The supernatantsamples were stored at -20° C. until they were required for IgG₁,monoclonal antibody concentration determination by an ELISA method. Oncompletion of the 12-15 day culture period all the cells were recoveredfrom the cell culture receptacle 28 (i.e. >95%) by expelling thecell/supernatant mixture with sterile air. Finally, the cell culturereceptacle 28 was flushed twice with fresh media. The recovered cellswere counted and viabilities were determined.

FIGS. 14 and 15 demonstrate the effect of rotation speed on bothantibody production and cell viability. It is evident that 60 cpmresults in a dramatic increase in productivity (i.e. in the range ofabout 100%), with no net increase in viable cells during the course ofthe experiment. In the case of biomolecule production 60 cpm ispreferred. Where the goal of a cell culture process is to expend andharvest viable cells, lower cpm values (i.e., 30 cpm, 12 cpm or evenstatic) would be preferred.

EXAMPLE II

A series of four lipofection-based gene transfection experiments wereconducted in the cell culture incubator of the present invention with anHPBr device being the cell culture receptacle. The control experimentwas carried out in parallel with its own control which was done inmultiwell plates in an incubator. The following experimental procedureswere employed.

Plate Experiment

SW480 P3 (ATCC # CCL228) colon carcinoma cells were plated in 6-well(i.e., 1×106 viable cells per well). Thirteen wells were plated in thismanner. Each well contained 3 mL of a stock solution comprised of 26.4mL RPMI media, 4 mM L-glutamine 3.0 mL Fetal bovine serum, and 10 μg/mLgentamicin to make a total of 30 mL. Cells were cultured at 37° C. in aCO₂ incubator with 10% CO₂ for 24 hours prior to transfection. The cellswere able to adhere to the plates during this period.

The transfection procedure was done by replacing the previous media in 1mL OPTI-MEM media and adding a mixture of cationic lipid (DMRIE/DOPE)plasmid DNA (VR1412), both by Vical, Inc., San Diego, Calif. A molarratio of 0.99 (i.e., 40 μL lipid: 10 μg DNA) diluted in 2.0 mL OPTI-MEMwas applied to each well. These plates were incubated for 4 hours.

After 4 hours, heat deactivated fetal bovine serum plus 12.0 μLgentamicin were added to each well. For the following 13 days all of thecells from one well were trypsinized, counted, and 2×10⁴ retained as alysed sample for β-galactosidase reporter gene determination. Theremaining wells were feed with 1 mL of the previously defined RPMI mediaevery other day (starting 24 hours after transfection), without removingthe DNA containing OPTI-MEM.

At the end of the 13 day period the lysate samples were assayed forβ-galactosidase via a chlorophenol red-based procedure, which werequantitated with a UV-visible spectrophotometer.

HPBr Device Experiments

Four β-galatosidase reporter gene transfection experiments wereconducted in four cell culture receptacles 28 that were HPBr-typedevices with an equivalent protocol to that previously described to6-well plates, except for the differences documented below. All featuresof the procedure were adjusted on a "per cell" basis. However, due tothe perfusion mode of cell culture which is characteristic of the HPBrdevice (i.e., continuous feeding), there was no requirement for periodicfeeding by hand.

Sufficient Cytodex 1 microcarriers (by Sigma, St. Louis, Mo.) wereintroduced into the side ports of the cell culture receptacle 28 afterpre-swelling in phosphate buffered saline to have about 10 cells permicrocarrier bead. 1×10⁷ SW480 viable cells injected.

Table I lists the different media conditions for the first 24 hoursafter inoculation and post-transfection. It also identifies the rotationparameter employed in this study. 0.1 mM chondroitin sulfate (type C)was included in the OPTI-MEM transfection media for run Nos. 2, 3, and4. While the recirculating OPTI-MEM media was replaced by transfection,the media in the compartment containing the cells was not.

Daily samples (about 1.5 mL) of cell/supernatant were taken from eachcell culture receptacle 28 and an equal amount of fresh media was usedto replace it. Cell count and viability was determined and 2×10⁷ viablecells were lysed and retained for β-galactosidase determination.

                  TABLE I    ______________________________________    RUN  TYPE    CONDITION      MEDIA COMPOSITION    ______________________________________    1.   Plate   CO.sub.2 Incubator  control!                                1 L RPMI (see above for                                composition)    2.   HPBr    30 cpm         839 mL RPMI; 10% fetal                                bovine serum; 4 mM                                L-glutamine; 10 g/mL                                gentamicin; 2.5 mM benzoate                                buffer; 0.1 mM chondroitin                                sulfate (also present in                                OPTI-MEM transfection                                media).    3.   HPBr    Static         Same as run #2.    4.   HPBr    30 cpm for first 48 hr.,                                Same as run #2.                 then static    5.   HPBr    Static  control!                                1 L RPMI (see above for                                composition).    ______________________________________

Table II contains data comparing the four perfusion device experimentswith the plate control. It is evident that an HPBr-type cell culturereceptacle 28 operated in the cell culture incubator of the presentinvention can be employed to scale-up gene transfection and harvestingof cells, which may have therapeutic applications. This system can alsobe utilized as any artificial organ so that the long-term expression ofthe foreign gene can be easily and realistically studied; in a wayequivalent to taking a biopsy from an intact organ in vivo. In thisspecific instance, the device is employed as a solid tumor model.

                                      TABLE II    __________________________________________________________________________                                                        TOTAL                                      % INCREASED       EXPRESSION               TOTAL #                EXPRESSION                                               ng/mL PER                                                        BASED ON       Experimental               CELLS  %      AREA UNDER                                      PER 2 × 10.sup.4                                               2 × 10.sup.4                                                        VIABLE CELLS AT    Run       Conditions               (13 DAYS)                      VIABILITY                             CURVE (cm.sup.2)                                      CELLS    AT DAY 13                                                        DAY    __________________________________________________________________________                                                        13    1  Plate Control               6.5 × 10.sup.6                      97%    70       --       0.084    33    2  30 cpm    1 × 10.sup.7                      86%    84       20%      0.120    65    3  Static  2.3 × 10.sup.7                      42%    105      50%      0.602    364    4  30 cpm/48 hr               7.3 × 10.sup.7                      77%    180      157%     0.357    1254       then static    5  Bioreactor               29.3 × 10.sup.7                      34%    76        9%      0.040    249       Control (Static)    __________________________________________________________________________

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A modular cell cultureincubator comprising, in interlocking relationship:(a) a planarrectangular base; (b) a dividing planar rectangular wall extendingvertically from the base with a lower edge sized to extend transverseacross the base; (c) a first corner segment comprising a pair ofrectangular walls connecting at right angles, and a rectangular partialceiling portion extending between the pair of walls, with a longer edgeof the ceiling portion connecting to an upper end of one of the pair ofwalls, and a shorter edge of the ceiling portion connecting to an upperedge of another of the pair of walls; (d) a second corner segmentcomprising a second pair of planar rectangular walls connecting at rightangles, one of said walls having a smaller surface area than the other;and (e) a rectangular cage comprising two spaced planar rectangular sidewalls connected by a rectangular planar roof piece, and a rectangularplanar backing piece; whereby the base, dividing wall, first cornersegment, second corner segment and cage cooperate to form a rectangularcell culture incubator comprising two compartments separated by thedividing wall.
 2. The modular cell culture incubator of claim 1, whereinconnections between the base, first corner segment, second cornersegment and cage permit attainment of a pressure above surroundingambient pressure within the modular cell culture incubator.
 3. Themodular cell culture incubator of claim 1, wherein the base, firstcorner segment, second corner segment, and cage are comprised of atransparent plastic material.
 4. The modular cell culture incubator ofclaim 1, further comprising gaskets interposed between the first cornersegment and second corner segment.